Use of modified lysozyme c to prepare medicinal compositions for the treatment of some serious diseases

ABSTRACT

Use of modified lysozyme c or its pharmaceutically acceptable salts and its industrial production for the preparation of medicinal compositions, more particularly oral, parenteral and topical, for prophylaxis and therapy of some serious diseases in mammalians.

[0001] This invention relates to the use of modified lysozyme c compoundor to its pharmaceutically acceptable addition salts and to itsindustrial production for the preparation of medicinal compositions,more particularly oral, parenteral and topical, for prophylaxis andtherapy of some serious diseases in mammalians.

[0002] It is known that natural lysozyme is an enzyme of peptidicorigin, widely present in many species of the animal and vegetai world,having in the different species comparable but not identical structureand enzymatic mechanism. The most known lysozyme is that present in thechicken egg (chicken egg-white lysozyme c). Lysozyme is widelydistributed in the mammalians body; it is present for example in saliva,in tears, in milk, in leukocites, in cervical mucus. In 1922 AlexanderFleming discovered a substance present in his own nasal mucus able tolysate some bacterial strains. This substance has been later onidentified as lysozyme (today better identified as human lysozyme c). In1963 Jolles and Canfield independently clarified the primary structureof lysozyme purified from chicken egg-white. In 1965 Phillips describesthe tridimensional structure of lysozyme, on the basis of the X-rayscrystallography. As from its discovery, natural lysozyme (more preciselythe chicken egg-white lysozyme c) has been used sometimes in medicinemainly for its anti-bacterial properties, but the retrospective analysisof the published studies has not even today fully clarified its specificmechanism of action and in some cases the experimental results seem tobe even contradictory (1,2,3).

[0003] Nowdays scientists think that the biological action of naturallysozyme c shall be divided in two different ways: a direct action andan indirect action. The direct action is determined by the degradationof the link (β, 1→4) between N-acetylmuramic acid andN-acetylglucosamine (4) of peptidoglycanes present in the cell membraneof bacteria, thus determining the lysis of the same (as consequence ofthis action it is frequently called “muramidase”). Generally Grampositive bacteria are more sensitive to natural lysozyme's action thanGram negative, probably because the latter ones present the externalmembrane, as further barrier. This direct action of lysozyme ispotentially important for its possible therapeutic use in prophylaxisand treatment of many bacterial diseases, alike faringitis,congiuntivitis, otitis, sinusitis, adenitis, uretritis, vaginitis,cistitis, substantially caused by microorganisms of Streptococci family.By contrast, the degradated mucopeptidic fragments of the cell membrane,produced by the direct action of lysozyme on the bacteria, seem to beable to elicit a not well defined process of immunological stimulation(indirect action of lysozyme).

[0004] Some authors have also recently described the capacity of chickenegg-white lysozyme c and of its derivatives to stimulate the productionof lymphocytes T present in the lymphatic tissue, associated tointestine (GALT), and in spleen (GALT-speen axis). However in literaturesome authors have also described the use of other specific lyticpeptides for the treatment of many diseases. In some recent patents thespecific combination of the administration of Cecropins and lysozyme hasbeen suggested, in order to increase the action of the two compounds. Infact some authors suggest that Cecropins are much more active in lysingthe cell, while lysozyme is more active in its complete degradation.

[0005] The near chemotherapeutic agents, like Cecropins, Sarcotoxins,Magainins, Lepidopterans, having the same physical properties with anα-elique structure and hydrophobic character, are proteins able tolysate not only the cellular membrane of bacteria (Listeriamonocitogenes and Brucella abortus), but also the cell membrane ofprotozoos (P. Falciparum) and viruses (Parainfluenza and HerpesSimplex), and of eukariotes cells infected with bacteria. By using anelectronic miscroscope it has been discovered that lytic peptidesproduce large pores into membrane of cells, determining their death. Bycontrast, healthy mammalian cells are not destroyed by the above lyticpeptides, due probably to the presence of citoskeleter, which is incontact with various points of the plasmatic membrane and consequentlyit permits to maintain the osmotic integrity of the cell.

[0006] Moreover also at low concentrations the lytic peptides shall beused as biological response modifiers, in order to stimulate the cellsproliferation of the immunological system and of the skin. The aboveaction is probably caused by the production of pores in the plasmaticmembrane, determining a flow of ions and of nutritive material into thecells, which stimulates the cells' growth.

[0007] Nevertheless, although many publications evidence the positiveaction of natural lysozyme (mainly chicken egg-white lysozyme c) in thetreatment of infections of various origin, the potential use of group clysozyme in clinics has been very limited until nowadays due to threemajor factors:

[0008] a) human lysozyme c (which is the type present in humans) isextremely difficult to be obtained in sufficiently large amounts fortherapeutic applications;

[0009] b) other lysozyme c types, which are obtainable from otherspecies, alike bovine and chicken egg-white, may present some negativeside-effects (the first one is possible allergic reactions and thesecond one is the potential risk of contamination of transmissiblebovine encephalopathies or TBE), which are considerably limiting theiruse in medicine;

[0010] c) lysozyme types, different from c, originating from otheranimal species are not suitable for medicinal purposes since theypresent a very low homology with human lysozyme c.

[0011] A table is reported wherein the homology between human lysozymeand lysozymes from other mammalians, however all of natural c type, isindicated: LYSOZYME ORIGIN HOMOLOGY % Egg (white) chicken 56.8% Rat76.4% Cow 82.3% Rabbit 82.3% Horse 50.8%

[0012] Although bovine lysozyme c shows an homology higher than that ofchicken egg-white, the latter one is the most used in the clinicalpractice due that it is very commonly available in suitable quantitiesat convenient prices. On the basis of the above introduction there is aconsiderable interest to further explore new possibilities of use ofnatural lysozyme c, of its possible derivatives and to produce medicinalpreparations, which permit to widen its use to some serious diseases inhumans and/or to utilize new and previously unexplored administrationroutes.

[0013] Despite many experimental researches on animals and recentpublications on lysozyme c, the medicinal applications of this compoundwhen administered orally remained limited to traditional pathologies,already well known to the experts.

[0014] However these studies seem to have achieved very limited andsometimes contrasting results.

[0015] Therefore the main purpose of present invention is to providemedicinal compositions containing a modified natural lysozyme ccompound, more particularly a monomethoxypolyethylene glycol lysozyme ccompound (hereinafter defined as <<MLc>> for practicalness of thedescription) or its addition salts with pharmaceutically acceptableacids, presenting the following general formula (I):

[CH₃O—(CH₂—CH₂—O)_(n)—CH₂—CH₂]_(x)—(NH)_(y)Lysozyme c. (Z)_(m)

[0016] wherein:

[0017] y=number of free amino groups of molecules of lysine present inthe molecule of lysozyme c, preferably comprised between 5 and 30;

[0018] x=number of molecules of monomethoxypolyethylene glycol for eachmolecule of lysozyme c, where the value may vary from 1 to y;

[0019] n=number of polymerization degree of polyethylene glycol portion,preferably comprised between 5 and 1000;

[0020] Lysozyme c=lysozyme c from chicken egg-white, rat, cow, rabbit,horse or human;

[0021] Z=pharmaceutically acceptable organic or inorganic acid,monovalent or polyvalent, able to produce addition salts with theconsidered lysozyme c;

[0022] m=number of molecules of pharmaceutically acceptable acid usedfor the obtention of MLc addition salt, which may vary from 0 to thevalue corresponding to total number of amino groups present in themolecule of lysozyme c.

[0023] This compound is surprisingly and particularly useful andeffective for the specific treatment of some serious diseases inmammalians.

[0024] More particularly in the modified lysozyme c compound, utilizedas active ingredient for the preparation of medicinal compositionsuseful for the treatment of some serious diseases in mammalians, thenumber of radicals of monomethoxypolyethylene glycol (x) may vary from 1to “y”, the polymerization degree (n) of the polyethylene glycol portionof monomethoxypolyethylene glycol shall be comprised between 5 (M.W.about 161) and 1,000 (M.W. about 32,206), more preferably between 120(M.W. 3,865) and 200 (M.W. 6,441), while the number of molecules of theamino acid lysine (y), having a —NH— radical linked withmonomethoxypolyethylene glycol, in lysozyme c may vary from 5 (human andchicken egg-white), to 11 (bovine) or even more, to a maximum of 30.

[0025] Z means any pharmaceutically acceptable organic or inorganicacid, mono or polyvalent, pharmaceutically acceptable, which may becombined in different fashion, being “m” the number of molecules of thepharmaceutically acceptable acid, which may be required to yieldaddition salt with MLc and which may vary from 0 to the valuecorresponding to the total number of amino groups present in themolecule of the considered lysozyme c, value which shall be divided bythe valence of the same acid (Z).

[0026] Recently some authors have published that generally proteins canproduce, by quite simple chemical reactions already known to theartisans skilled in the art, derivatives of condensation withpolyethylene glycols.

[0027] Therefore also the chicken egg-white lysozyme c may produce, byreacting with monomethoxypolyethylene glycol (polymerization degree ncomprised between 5 and 1000) or with its activated derivative(tresylated monomethoxypolyethylene glycol), with a reaction providing adirect linkage between the polymer and the protein via a stablesecondary amine, the pegilated derivative of chicken egg-white lysozymec.

[0028] At the present state of the art pegilated derivatives of otherlysozymes of c group are unknown, and therefore they shall be considerednot described and novel, as also their therapeutic use.

[0029] It has been also observed that these pegilated derivativespresent generally a reduced immunoreactivity and a better stabilityagainst heating and degradation during their storage. Some authors havealso formulated the hypothesis that these pegilated derivatives couldshow, after their absorption, also a better resistance to theproteolytic agents and therefore a better half-life in plasma.

[0030] These pegilated derivatives can be also obtained by a synthesisprocess, which determines a covalent binding of the selected naturallysozyme c with the selected monomethoxypolyethylene glycol radicals,after exposing at room temperature during about 2 hours lysozyme c totresylated monomethoxypolyethylene glycol in PBS (Phosphate BufferSaline) or in a borate buffer at different pH (almost always in slightlybasic medium) and in different proportions. The tresylated glycolreagent may be commonly obtained from monomethoxypolyethylene glycolactivated with tresyl chloride. Monomethoxypolyethylene glycol isselected to provide only one reactive end in each molecule, thusavoiding undesired cross-linked products, but yielding only the desiredMLc selected compound. The purification of MLc is carried out usingShepadex G-50 columns (12 cm×1.3 cm) stabilized with PBS, or byultracentrifugation with Amicon membranes of desired porosity. In caseit is desired to obtain the addition salts of MLc, for example MLchydrochloride, it is necessary to carry out a dialisis with an aqueoussolution of diluted HCl (10-100 mM) for all night at 4° C. or morepreferably for 6-8 hours at room temperature, then carrying out thepurification by Sephadex G-50 column. The resulting salt shall be thenlyophilized and stored at temperatures below 0° C., util to be used.

[0031] A possible general synthesis scheme of addition salts of MLc areindicated herebelow, for a better understanding of their synthesisprocess (NOTE: the amino groups of lysozyme c are evidenced for a betterunderstanding):

[0032] A=monomethoxypolyethylene glycol (MPEG) with only one finalreactive group;

[0033] B=tresyl chloride (2,2,2-trifluoroethane-sulfonyl-chloride);

[0034] C=tresylated monomethoxypolyethylene glycol (TMPEG);

[0035] D=lysozyme c;

[0036] E=modified lysozyme c (MLc);

[0037] F=pharmaceutically acceptable acid;

[0038] G=MLc addition salt.

[0039] Protein concentrations are then measured directly by determiningthe absorbance at 280 nm and by “Coomassie brilliant blue” titration.For the colorimetric reaction 100 μl of the protein sample is aliquotedin microtiter plates and 200 μl of “Coomassie protein assay” reagent areadded. The absorbance at 555 nm is measured by means of a microtiterreader after 5 minutes of incubation at room temperature. By using boththe described methods, the concentration of the samples is establishedby means of a calibration curve obtained from a stock solution oflysozyme c at a known concentration.

[0040] The obtained MLc is then tested by determining the assay usingthree different methods: 1) SDS-Polyacrylamide gel electrophoresis; 2)Molecular filtration chromatography; 3) Enzymatic activity according tothe conventional method.

1) SDS-Polyacrylamide Gel Electrophoresis

[0041] A 15% polyacrylamide gel is used. The running is usually carriedout between 100 and 150 V, for about 45 minutes.

[0042] Concentrated solution of acrylamide (30%)/bisacrylamide:

[0043] Dissolve 29.2 g of acrylamide and 0.8 g of bisacrylamide in 70 mlof deionized water. Once acrylamide is dissolved, add water to yield avolume of 100 ml. Filtre the solution through a 0.45 μm pore sizemembrane. The obtained solution is stable at 4° C. for 1 month whenstored in a dark bottle.

[0044] Buffer A (1.5 M Tris-HCl buffer, pH 8.80):

[0045] Dissolve 18.2 g of Tris base in about 80 ml of distilled water.Adjust the pH to 8.80 with HCl and yield 100 ml volume adding distilledwater. Store the obtained Buffer A at 4° C.

[0046] Buffer B (0.5 M Tris-HCl buffer, pH 6.80):

[0047] Dissolve 6.1 g of Tris base in 80 ml of distilled water. Adjustthe pH to 6.80 with HCl and yield 100 ml volume adding distilled water.Store the obtained Buffer B at 4° C. 10% (w/v) sodium dodecylsulphate(SDS):

[0048] Dissolve 10 g of SDS in 60 ml of water. Bring to 100 ml volumewith distilled water.

[0049] Dissolution buffer of the sample (0.06 M Tris-HCl, pH 6.80. 2%SDS, 10% glycerol, 0.025% bromophenol blue): Water 4.80 ml Buffer B 1.20ml SDS 10% 2.00 ml Glycerol 1.00 ml Bromophenol blue 0.5% 0.50 ml

[0050] Store the above solution at room temperature. The SDS-reducingbuffer is prepared adding 50 μl of 2-mercaptoethanol to each 0.95 ml ofbuffer solution of the sample. Prepare this solution at the moment ofits use.

[0051] Catalyst (10% w/v ammonium persulphate):

[0052] Dissolve 100 mg of ammonium persulphate in 1 ml distilled water.Prepare this solution at the moment of its use.

[0053] TEMED:

[0054] Use directly the product present in the bottle. Store in a notwet place, protected from light.

[0055] Elution buffer (0.025 M Tris, 0.192 M glycerol, 0.1% (w/v) SDS pH8.30±0.20):

[0056] Dissolve 0.3 g of Tris base, 1.4 g of glycerol, 1 ml of 10% SDSin 100 ml distilled water. Store this elution buffer at 4° C.Preparation of 15% polyacrylamide gel (0.375 M Tris, pH 8.80): Distilledwater 2.35 ml Buffer A 2.50 ml 10% SDS (w/v) 0.10 ml Concentratedsolution of: 30% acrylamide/bisacrylamide 5.00 ml 10% (w/v) ammoniumpersulphate 0.05 ml TEMED 0.005 ml Total volume 10.005 ml Preparation of4% polyacrylamide gel (0.125 M Tris, pH 6.80): Distilled water 6.10 mlBuffer B 2.50 ml 10% SDS (w/v) 0.10 ml Concentrated solution of: 30%acrylamide/bisacrylamide 1.30 ml 10% (w/v) ammonium persulphate 0.05 mlTEMED 0.005 ml Total volume 10.055 ml

[0057] Sample preparation:

[0058] Dilute the sample with 4 volumes of reducing-SDS buffer and heatto 95° C. for 4 minutes (each band of the gel shall contain not lessthan 1 μg of protein). The electrophoretic migration is carried out byusing a MiniProtean II (BioRad, California) equipment, at roomtemperature. The applied voltage is 100-150 V for about 45 minutes.

[0059] Gel coloration with Coomassie brillant blue R-250:

[0060] Dissolve 0.2 g of Coomassie brillant blue R-250 in 1 litre of asolution containing 50% (v/v) of methanol, 10% (v/v) of acetic acid and40% (v/v) of distilled water. Filtre the solution using a filteringpaper and store the solution in an ambered bottle. Before using theobtained solution, it is necessary to check always if a colouredprecipitate is present. In affermative case filtre again.

[0061] Place the gel during 10 minutes in a solution containing 50%(v/v) of methanol, 10% (v/v) of acetic acid and 40% (v/v) of distilledwater in order to eliminate the presence of SDS.

[0062] Keep the gel in the coloured solution under constant and slowstirring during 30 minutes at 50° C. or alternatively during two hoursat room temperature.

[0063] Preserve the gel in a liquid medium consisting from a 10% (v/v)acetic acid solution. Alteratively dry the gel using a glycerol solutionand a cellophane film and proceed in the following way:

[0064] a) place the gel for 10-15 minutes in a 10% (v/v) glycerolsolution; b) wet the cellophane film in the same solution: c) place thegel between two wetted cellophane films without introducing air bubbles;d) place the gel on a shelf and leave it at room temperature.

[0065] Evaluation of the results:

[0066] The sample composition is determined by densitometry at 595 nm onthe gel, using a suitable equipment, as for example Shimadzu UV/VIS CS930 scanner.

[0067] Acceptability limit:

[0068] The sample shall contain a single spot, after coloration byCoomassie brillant blue R-250.

[0069] MLc content shall not be less than 95%.

2) Molecular Filtration Chromatography

[0070] The sample is chromatographied using a Superose 12HR 10/30 columnand an equipment for FPLC (Fast Performance Liquid Chromatography)equilibrated with PBS. The sample is diluted with PBS before loading iton the column. Inject 200 μl of the sample and elute with PBS at a flowrate of 0.3 ml/min. Collect fractions of 0.25 ml. The elution profile isdetermined by spectrophotometry at 280 nm. The proteic concentration isdetermined quantitatively by using Coomassie brilliant blue.

[0071] Use markers with known molecular weight at a concentration of 3mg/ml, as for example β-amilase (200 KDa), alcohol dehydrogenase (150KDa), bovine serum albumine (66 KDa), carbonic anhydrase (29 KDa),citochrome C (12.4 KDa), aprotinine (6.5 Kda). MLc elutes at about 17.35ml under the indicated conditions.

[0072] Acceptability limit:

[0073] MLc content shall not be less than 95%.

3) Enzymatic Activity

[0074] This method is carried out according to the conventional methodsalready described in literature (5, 6, 7).

[0075] Test conditions:

[0076] A) Suspension of Micrococcus luteus (Micrococcus lysodeikticus)at a concentration of 0.3 mg/ml in 0.2 M phosphate buffer (pH=7.00) and17 mM of NaCl.

[0077] B) Solution of MLc (1 mg/ml) in distilled water.

[0078] C) Spectrophotometer suitable to determine absorbance variationsat 450 nm.

[0079] Procedure:

[0080] Place 2.9 ml of a Micrococcus luteus (Micrococcus lysodeikticus)suspension in a spectrophotometric cuvette. Leave the suspension for 4-5minutes at 25° C., in order to allow the suspension to reach thereaction temperature. Add 0.1 ml of enzymatic solution and record theabsorbance variation each minute at 450 nm.

[0081] Calculation of enzymatic activity of MLc:${{Units}\text{/}{mg}} = \frac{\left( {\Delta \quad {A_{450\quad {nm}}/\min}} \right) \times 10^{3}}{{mg}\quad {of}\quad {enzyme}\quad {in}\quad {the}\quad {reaction}\quad {mixture}}$

[0082] Enzymatic unit is defined as the enzyme quantity, which in thetest conditions (pH=7.00 at 25° C.) decreases of 0.001 each minute theabsorbance of Micrococcus luteus suspension. Therefore the abovedemonstrates that MLc maintains also the main enzymatic activity,already known in natural lysozyme c.

[0083] In another embodiment of the instant invention the authors havesurprisingly noticed that the administration route of MLc or itspharmaceutically acceptable salts is fundamental and determinant inorder to elicit unknown and remarkable therapeutic effects on somesevere diseases in mammalians.

[0084] In fact the authors have surprisingly discovered that oraladministration of MLc may induce high plasmatic levels of free TNF-α(Tumor Necrosis Factor alpha), biologically available, thus inhibitingby a practically direct mechanism and significantly the replicationspeed of the tumoral proliferative process in mammalians (8, 9, 10).

[0085] In fact an embodiment particularly preferred of the presentinvention is the use of MLc for the preparation of medicinalcompositions to be administered by oral route or, that in another way,the oral administration of MLc is suitable to induce a significantplasmatic increase of free TNF-α, resulting this last substance, whenbiologically available, particularly useful for its protective effectand for the specifc treatment of tumoral processes in general,expecially when all at the initial stage.

[0086] It is in fact known from the most recent publications that TNF-αis a specific factor, as indicated by its name, able to induce theinhibition, however the necrosis, of the tumoral process of the cellsalso when grown in vitro.

[0087] The tumoral processes are paradoxically accompanied by highhaematic levels of TNF-α, which is linked to soluble forms of receptorsand therefore it is not free and biologically available. On the contraryit is very important to induce high levels of free TNF-α (11, 12, 13).The mechanism why MLc only by oral administration is able to remarkablyinduce the increase of the plasmatic levels of free TNF-α is stillunknown, being still matter of research by the authors, but it can behypotethically related in vivo to a possible active stimulation of thebiosynthesis process of TNF-α. The mechanism can be explained in the waythat the gene transcription of TNF and mRNA translation are bothstrongly stimulated by peptidoglycans, which are the most direct andsignificant expression of the enzymatic and lytic activity of MLc,administered by oral route, on cell walls of bacteria, normallycomponents of intestinal flora (14), thus demonstrating by contrast thatMLc has maintained its enzymatic activity similar to that correspondingto natural lysozyme c, which is in net contradiction with thepublication of other authors. Concerning the mechanism it is necessaryto consider that a recent study has indicated that MAP kinase homologsbecome phosphorylated in peptidoglycans-stimulated cells, thussuggesting their possible involvement in signal transduction.

[0088] Most of the pleiotropic biological actions of TNF can beattributed to its ability to activate a variety of genes in a multitudeof target cells. TNF-α and the functionally related cytokine IL-1 arethe only natural substances known to have such a large spectrum oftarget gene. Partial list of proteins induced by TNF includes: IL-1α andIL-1β, IL-6, IL-8, IFN-β, IL-2 receptor α chain and many others.

[0089] Surprisingly the authors have also noted in the instant inventionthat the oral administration of MLc decreases also the plasmatic levelsof sialic acid (total), which is an “aspecific marker”, which increasespathologically in some serious degenerative processes, includingdifferent types of tumors. The above finding further supports that freeTNF-α stimulation, obtained from oral administration of MLc, determines,as claimed, therapeutic inhibition effects of neoproliferativeprocesses, which are expression of tumoral cells in animals. Thereforeone may conclude that the first objective of the present invention isthe use of MLc or its pharmaceutically acceptable salts for thepreparation of medicinal compositions to be administered only by oralroute in order to obtain a remarkable increase of plasmatic level offree TNF-α, thus biologically available, and consequently particularlyuseful in the prophylaxis and treatment of tumoral processes inmammalians, for which an increase of plasmatic free TNF-α (not blockedby soluble forms of receptors) is able to produce beneficial andtherapeutic effects.

[0090] Other preferred and important embodiment of the present inventionis the use of MLc or of its pharmacologically acceptable salts for thepreparation of medicinal compositions particularly useful for thetreatment of rheumatoid arthritis, Acquired Immuno Deficiency Syndrome,more commonly known with the Anglo-Saxon terminology “AIDS”, psoriasisand septic shock, where the most preferred administration route, toelicit the desired pharmacological and therapeutical effects, is theparenteral route, more precisely the intravenous route. In fact it hasbeen experimentally supported that, when MLc is administered byparenteral route, more preferably by intravenous route, stronglyinhibits the TNF-α plasmatic levels, thus surprisingly inducing apharmacological response, which is just opposite to that achieved whenMLc is administered by oral route.

[0091] The main target of the chronic inflammatory response inrheumatoid arthritis (RA) is the synovium. Even though it is unknownwhat causes this desabling disease, characterization of the infiltratedinflammatory cells and their products is of great importance tounderstanding its pathogenesis.

[0092] There is considerable evidence involving TNF-α in thepathogenesis of RA. This evidence is based not only on the generalizedpresence of TNF-α in arthritic joints, accompanied by an increasedresponse of TNF-α receptors, but also from the effects of aneutralization of TNF-α in joint cells cultures.

[0093] Thus neutralization in vitro of TNF-α elicits the inhibition ofthe production of interleukin 1, as TNF-α, which is believed to be thecause determining the inflammation and the erosion of the joint.

[0094] In AIDS disease TNF-α is a potent activator of the replication ofthe HIV virus in the infected cells. In symptomatic patients the levelsof TNF-α are higher than in asymptomatic patients. Probably TNF-αdetermines a transcriptional activation of HIV provirus integrated inthe genoma of the host cell, by increasing the levels of viral RNA.Moreover TNF-α contributes to the viral replication, which increases inturn the destruction of CD₄ ⁻ limphocytes and reduces the immunologicalfunctions of the body (15).

[0095] Consequently in the pathologies, as RA but especially AIDS,wherein the levels of TNF-α are high, the administration of MLc by oralroute is not suitable, but absolutely self-defeating andcontra-indicated because it may cause an exacerbation of the disease,increasing the TNF-α levels which are already very high. By contrast thepresent invention has surprisingly evidenced that the parenteraladministration of MLc or of its pharmaceutically acceptable salts, moreparticularly by intravenous route in AIDS and intra-articularly in R.A,induces a remarkable and favourable therapeutic decrease of TNF-α levelsin fluids (plasma in AIDS and sinovial fluid in R.A.). This importanttherapeutic effect, subject matter of the present invention, supportsthe hypothesis that the decrease of TNF-α may be attributed from thereflected inhibitory action (“rebound” effect) on cellular organs or onthe mechanism at the origin of these pathologies.

[0096] Moreover an experimental significant reduction of TNF-α in plasmaand in cerebrospinal fluid (CSF) were observed in non controlled studiesin humans, and therefore the therapeutic indications of the medicinalparenteral preparations of MLc or its pharmacologically acceptablesalts, are also particularly extended to reverse severe Central NervousSystem impairment (“AIDS dementia complex” and other correleted seriousmotorial and cognitive disorders).

[0097] Another particularly preferred embodiment of the presentinvention is the use of MLc or of its pharmacologically acceptable saltsfor the preparation of a topical medicament, capable to elicit aremarkable antihistaminic activity, when MLc or its pharmaceuticallyacceptable salts are locally applied. These topical medicinalpreparations of MLc or its pharmacologically acceptable salts have shownin experimental non controlled studies to prevent blood flow increase,vascular permeability, edema, pain and irritation caused by the actionof histamine.

[0098] Another particularly embodiment of the present invention is thatthe topical medicinal preparations of MLc or of its pharmacologicallyacceptable salts result particularly active to determine also bytransdermic route the reduction of TNF-α levels, resulting the abovemedicinal products particularly indicated for the treatment of reactivearthritis, rheumatoid arthritis, psoriasis arthritis and generally inthose cutaneous or subcutaneous conditions where a reduction of TNF-αlevels is therapeutically required.

[0099] Another preferred embodiment of the present invention is that thetopical preparations of MLc or its pharmacologically acceptable saltsare particularly active in the topical treatment of acne and in theprevention of decayed teeth.

[0100] Another preferred embodiment of the invention is the use of MLcor its pharmaceutically acceptable salts for the preparation ofmedicinal products, wherein they may be associated, in differentquantities and proportions, to other substances with antibactericaction, from chemical and antibiotic origin, alike semi-synthetic orsynthetic penicillins, cephalosporins, macrolides, erytromycines,quinolonic derivatives, but just to mention some of the most importantones. These associations, conveniently formulated in suitable galenicalpreparations known to the artisans skilled in the art, are particularlyuseful to overcome infections or pathologies caused from bacteria,macrovirus, virus or prions, which are particularly sensitive to thesynergic therapeutic effect of these associations of MLc or itsderivatives.

[0101] Another preferred embodiment of the instant invention is that innon controlled studies, wherein for ethical reasons only a limitednumber of healthy volunteers for each indication has been admitted, themedicinal preparations of MLc or its pharmacologically acceptable saltsshowed surprising therapeutic effects, which cannot be explained by thepresent biological knowledge, but which will be matter of furtherresearches, on other serious pathologies, alike anorexia, multiplesclerosis, amiotrophic lateral sclerosis, Alzheimer disease, Parkinson,Bovine Spongiform Encephalopathy (BSE), Bovine TrasmissibleEncephalopathy (TBE), Creutzfeldt-Jacob diseases, miocardic infarct,ictus, all phatologies wherein nowdays a possible involvement ofbacteria, viral agents and/or infective proteic agent (prions) may beonly supposed in their etiophatogenesis.

[0102] The present invention also provides a suitable pharmaceuticalcomposition containing MLc or its pharmacologically acceptable salts,alone or associated with one or more active ingredients of the samegroup or with any other different medicinal substance or with a mixturethereof, prepared by using inert pharmaceutically acceptable carriers ordiluents, or a mixture thereof, at solid, liquid or gas state, at roomtemperature.

[0103] The pharmaceutical composition of the invention containing MLc orits pharmacologically acceptable salts, may be any preparations suitablefor oral route, alike tablets, with prompt or sustained release,capsules, granulate, powder, sirup, suspension or emulsion, drops, andalso suitable for parenteral administration, as sterile aqueoussolutions, sterile conditionned or lyophilized powder for extemporaneouspreparation of sterile injectable solutions or dispersions and finallyalso in the form of topical preparations, alike cream, ointment, lotion,gel and medicated gauze, and transdermic plaster. The pharmaceuticalcomposition of the invention containing MLc or its pharmaceuticallyacceptable salts may also comprise by systems for application byendonasal route, spray under pression or inhaler, and rectal and vaginalsuppositories, tooth-paste, all prepared according to general techniqueswell known to the skilled artisans.

[0104] In the medicinal compositions of the invention, to beadministered orally, MLc or its pharmaceutically acceptable salts aremixed alone or with other active ingredients, in suitable quantities andproportions, in a convenient pharmaceutically acceptable carrier, solidor liquid or a mixture thereof so that to produce suitablepharmaceutical forms containing the desired doses. The oral dose unitpreferably contains from 5 to 80% of the active ingredient, morepreferably between 40% and 60%.

[0105] Some conventional suitable inert solid carriers are for examplelactose, starch, talc, microcrystalline cellulose, calcium phosphate,magnesium stearate, dextrins and/or other excipients, binders,disintegrants, diluents, lubricants, sweetening and flavouring agents,already known in the art, with or without the use of water or of anothersuitable auxiliary solvent.

[0106] Conventional aqueous solutions suitable for oral use of MLc orits pharmacologically acceptable salts can be prepared by dissolving theactive ingredient in water or in any other suitable liquid solvent atroom temperature and adding different quantities and proportions ofsuitable colorants, sweeteners, flavours, stabilizers, surfactants andthickening agents, as desired.

[0107] Aqueous suspensions suitable for oral use can be prepared bydispersing the finely powdered MLc or its pharmaceutically acceptablesalts in water or in any other pharmaceutical suitable carrier withsuspending agents, as natural or synthetic gums, resins,methylcellulose, sodium carboxymethylcellulose and others well known inthe art.

[0108] The parenteral preparations of the invention, containing MLc orits pharmaceutically acceptable salts, may be formulated according tothe known art, using suitable, parenterally acceptable solvents ordiluents, more particularly water with other auxiliary excipients alikesodium chloride, sugars, buffers, ethanol, polyols (glycerol, propyleneglycol), preserving agents and absorption delaying agents.

[0109] The use of these agents for suitable pharmaceutically acceptablecompositions of MLc or its pharmacologically acceptable salts, isalready well known in the art. In fact in the medicinal compositions ofthe invention any conventional excipient or a misture thereof may beincluded, except insofar, as any conventional media or agentincompatible with the active ingredient.

[0110] It is especially convenient to formulate the parenteralcompositions of the invention in unitary dose form to simplify itsadministration and to achieve uniformity of dosage, alike ampoules orvials to be produced, according to the techniques already known to theskilled artisans, under sterile and aseptic conditions and packed inatmosphere of an inert gas (nitrogen) to prevent possible degradationprocesses.

[0111] The concentration of active ingredient, as solution or as powderto be reconstituted, may vary from 0.5% to 10%, preferably from 2% to4%. The preparations of the invention for topical or local use, areproduced with conventional techniques already known to skilled artisans,suitable for the preparation of these pharmaceutical forms to be appliedlocally on the skin at therapeutically effective dosages.

[0112] When the medicinal preparations of the invention are used fororal administration, each oral unit dose may contain a quantity of MLcor its salts varaying from 50 mg to 2000 mg, preferably from 250 to 1000mg (or every 5 ml in case of liquid forms), while in case ofpreparations to be used parenterally the unit dose may contain from 100mg to 2000 mg in 50 ml, preferably from 500 mg to 1000 mg, independentlythat the active ingredient is already dissolved in a solution orconditionned or lyophilized powder to be reconstituted with 50 ml ofsolvent. For topical preparations of the invention the content of thecompound of formula (I) may be used at a variable concentrationcomprised between 0.5% and 10%, preferably between 1% and 5% of thetotal content.

[0113] When the oral administration is adopted, therapeutic daily dosesof MLc or its salts comprised between 150 mg and 6 g a day, morepreferably comprised between 500 mg and 2.5 g a day, are generally used.By parenteral route the total daily doses of MLc or its salts may varyfrom 200 mg to 3 g daily, preferably between 500 mg and 2 g daily.

[0114] These dosages may be increased in relation to the pathology totreat, to its seriousness level and to the desired therapeutic response.Also the duration of the treatment may vary considerably, always inrelation to the desease to treat, to its seriousness and to thetherapeutic response which is obtained during the treatment.

[0115] However it is understood that the specific dosage, the dosagefrequency, the total daily dose, and overall the length of treatment canbe subject to significant variations in each patient depending on avariety of factors and evaluation criteria, alike activity of thespecific compound, seriousness and pathology to treat, age, body weight,general health, sex, diet, rate of excretion, drug combination, generalconditions of person undergoing therapy.

References

[0116] (1) Ossermann. E. F. Klockars. M., Halper J. e Fischel, R. E.(1973). Effects of lysozyme on normal and transformed mammalian cells.Nature 243: 331-335.

[0117] (2) Rinehart, J., Jacobs. H. e Ossermann. E. (1979). Lysozymemodulation of lymphocyte proliferation. Clin. Res. 27: 305A.

[0118] (3) Lemarbre. P., Rinehart. J. J., Kay N. E., Vesella, R. eJacobs, H. S. (1981). Lysozyme enhances monocyte-mediated tumoricidalactivity: a potential amplifying mechanism of tumor killing. Blood 58:994-999.

[0119] (4) Chipman. D. M. e Sharon, N. (1969). Mechanism of Lysozymeaction. Scienze 165: 454-465.

[0120] (5) Shugar D. (1952). Measurement of lysozyme activity and theultra violet inactivation of lysozyme. Biochim. Biophys. Acta 8:302.

[0121] (6) Minamiura N., Yamamoto T., Fukomoto J. (1966) Agr. Biol.Chem. 30:186;

[0122] (7) Malamy M., Horecker B. L. (1964) Biochemistry 3:1894.

[0123] (8) Sava, G. Perissin, L., Zorzet, S. e Callerio C. (1986).Antineoplastic effects of egg-white lysozyme in mice bearing solidmetastasizing tumors. Anticancer Res. 6: 183-186.

[0124] (9) Sava, G., Ceschia, V. e Zabuchi, G. (1988). Evidence forhost-mediated antitumor effects of lysozyme in mice bearing the Mcamammary carcinoma. Eur. J. Cancer Clin. Oncol. 24 (11): 1737-1743.

[0125] (10) Sava, G., Ceschia, V K. e Pacor, S. (1989). Mechanism of theantineoplastic action of lysozyme evidence for host mediated effects.Anticancer Res 9: 1175-1180.

[0126] (11) McIntosh, J. K., Mule, J. J., Travis, W. D. and Rosemberg,S. A. (1990). Studies of effects of recombinant human tumor necrosisfactor on autochthonous tumor and transplanted normal tissue in mice.Cancer Res. 50: 2463-2469.

[0127] (12) Semenzato. G. (1990). Tumor necrosis factor: A cytokine withmultiple biological activities. Br. J. Cancer 61: 354-361.

[0128] (13) Blankenstein. T., Qin, Z. H., Sberla, K., Moller, W., Rosen,H., Volk, H. D. et al. (1991). Tumor suppresion after tumorcell-targeted tumor necrosis factor alpha gene transfer. J. Exp. Med.173: 1047-1052.

[0129] (14) Namba, Y., Hidaka Y., Taki. K. e Morimoto, T. (1981).Effects of oral administration of lysozyme or digested cell walls onimmunestimulation in guinea pig. Infect. Immunol. 31: 580-583.

[0130] (15) Vilcek. J. and Lee, T. H. (1991). Tumor necrosis factor. Newinsights into the molecular mechanisms of its multiple actions. J. Biol.Chem. 266 (12):7313

[0131] The invention is further described for a better understanding inthe following Examples, which shall not be considered limitative of thesubject matter and of claims of the instant invention.

EXAMPLE 1

[0132] Evaluation of serum levels of cytokines. TNF-α, IL-1α and IL-1β,IL-6 and IL-8, in CBA mice following to oral administration of MLc(chicken egg-white) hydrochloride for 7 consecutive days.

[0133] The CBA strain mice treatment was performed by oraladministration of daily dosages of MLc hydrochloride (chicken egg-white)at 25-100 mg/Kg/day for 7 consecutive days (quantity expressed asequivalent of lysozyme c of chicken egg-white). The compound undertesting was carefully admixed with the powdered food. The level ofTNF-α, IL-1α, IL-1β, IL-6, IL-8 has been determined on day 7 by enzymeimmunoassay (ELISA). Table 1 shows the obtained results, evidencing theincreased level of plasmatic TNF-α and consequently of other cytokinesfollowing after administration of MLc (chicken egg-white): TABLE 1PLASMATIC LEVELS OF TNF-α AND CYTOKINES (pg/ml) Following 7 days oraltreatment with MLc (chicken egg-white) Pre-Treatment (mg/Kg/day) TNF-αIL-1α IL-1β IL-6 IL-8 0 6.14 7.16 1.51 3.52 2.81 25 11.14 10.74 2.425.24 4.50 100 11.80 11.07 2.57 5.63 4.06

EXAMPLE 2

[0134] Evaluation of serum levels of TNF-α and IFN-α (alpha-interferon)in volunteer patients with AIDS following administration of MLc (chickenegg-white) hydrochloride by intravenous route (phleboclysis). It hasbeen confirmed that in AIDS, TNF-α and TNF-β appear to play a crucialrole, by enhancing the replication of HIV, as well as inducing their ownexpression and that of other cytokines. Plasmatic levels of TNF-α andIL-1 are substantially increased during AIDS progression. Consequentlygaining a control over the TNF-α induction, it is possible to establishan effective therapy for AIDS.

[0135] MLc as it is or as salt inhibits the synthesis of TNF-α andstimulates simultaneously the synthesis of IFN-α.

[0136] In view of the above considerations 20 mg/Kg/day of MLchydrochloride (quantity expressed as equivalent of chicken egg-whitelysozyme c) was administered by slow intravenous route to healthycontrols, HIV-infected patients and HIV-infected asymptomatic patientsduring “n” consecutive days (n=not less than 120 days). Table 2 showsthe obtained results, confirming a reduction of TNF-α plasmatic leveland an increase of IFN-a following administration of MLc (chickenegg-white) hydrochloride: TABLE 2 TNF-α IFN-α Number Plasmatic PlasmaticControl 20 10.30 1.60 HIV-infected patients symptomatic 15 20.09 <1asymptomatic 10 9.35 0.98 HIV-infected symptomatic patients + 9 11.303.57 +MLc (chicken egg-white) hydrochloride

EXAMPLE 3

[0137] Study on the effect of intravenously administered MLc (rat)acetate in DBA/1 mice with collagen-induced arthritis. DBA/1 mice werechosen because of its many immunological and pathological similaritiesto human RA (rheumatoid arthritis). MLc (rat) acetate at the dose of 20mg/Kg of body weight (quantity expressed as equivalent of rat lysozymec), was injected intraveneously to adult mice both before the inductionof the arthritis or after the onset of the disease. MLc (rat) acetate,administered by parenteral route prior to disease induction, determinesa significant reduction of pain and of histological severity ofarthritis. By comparing the targets of the study to the analog humandisease, the capacity of the selected product to reduce clinicalconditions, joint swelling, and histological severity of disease, evenwhen administered after the onset of clinical arthritis signs, is ofgreat significance. Mononuclear cells from RA joints maintained inculture produce many cytokines with pro-inflammatory activity, includingTNF-α, measured by Elisa tests. MLc acetate in vitro reduces theproduction of this pro-inflammatory cytokine. Mononuclear cells (106cells per test) were activated with PHA at the concentration of 1 μg/mlas from day 1st to 5°. Then the cells were washed three times with PBSand cultured in complete medium (500 μl) in presence or absence of MLc(rat) acetate. After two days the culture supernatant was removed andtested for its content of cytokines. Table 3 confirms the decrease ofTNF-α levels after intravenous administration in those lots previouslytreated with MLc (rat) acetate. TABLE 3 CYTOKINE LEVELS (pg/ml)Treatment TNF-α IFN-α IL-1α Without MLc (*) 301.60 7.30 2.51 +MLc (*)0.1 mg/ml 55.79 9.76 3.42 +MLc (*) 1 mg/ml 20.60 18.07 3.57

EXAMPLE 4

[0138] Preparation of 100,000 tablets containing 300 mg of MLchydrochloride (chicken egg-white).

[0139] Each tablet is containing: Ingredients Quantity (per tablet) MLchydrochloride 300 mg (chicken egg-white) Magnesium stearate 25 mgMicrocrystalline cellulose 250 mg Silicon dioxide 15 mg Sodiumpolycarboxymethylcellulose 20 mg Total weight 610 mg

[0140] The manufacturing process is carried out by direct compression ofthe powder mixture through the steps already known to skilled artisansto prepare tablets, using suitable rooms and equipments for this type ofproduction.

[0141] 1) 30 Kg of MLc hydrochloride (chicken egg-white) and 25 Kg ofmicrocrystalline cellulose are placed into a suitable blender, afterpassing through a seive of 1.2 mm mesh; 2) to the powder mixturepreviously prepared 2.5 Kg of magnesium stearate, 1.5 Kg of silicondioxide and 2.0 Kg of sodium polycarboxy-methylcellulose are added inthe minor possible time in order to obtain the content uniformity; 3)the obtained mixture is stored temporarily in suitable containers, andafter approval by quality control of the active ingredient content, isthen transferred to the compression unit, wherein the powder isconverted into tablets by direct compression, by using rotary machinewith a suitable punch of the desired measure.

[0142] 97,931 biconvex tablets containing each the desired quantity ofMLc hydrochloride are obtained. Effective yield (97,931 tablets) 97.93%(Theoretical yield 100,000 tablets 100.0%) Effective mean weight 612.50mg (Theoretical mean weight 610.00 mg) Effective mean content for eachtablet 301.60 mg of MLc hydrochloride (Theoretical content for eachtablet 300.00 mg of MLc hydrochloride)

EXAMPLE 5

[0143] Preparation of 1,500 ampoules of 10 ml (15,000 ml) containing MLchydrochloride (chicken egg-white) (concentration of 50 mg/ml of MLchydrochloride).

[0144] 1 ml of solution is containing 50 mg of MLc hydrochloride.Ingredients Quantity (per ml) MLc hydrochloride (chicken egg-white) 50.0mg Sodium chloride 8.0 mg Water for injectable preparation q.s. to 1.0ml

[0145] The manufacturing process is carried out through steps alreadyknown to skilled artisans for the preparation of sterile and apyrogensolutions, using suitable rooms and equipments for the production ofinjectable solutions.

[0146] 1) In a stainless steel dissolutor about 12.0 K of water forinjections are introduced and under constant and slow stirring 120.0 gof sodium chloride are added; 2) separately in about 4.0 Kg of solution,prepared at the previous point, 0.750 Kg of MLc hydrochloride aredissolved; 3) solution at point (2) is added to solution (1) and thetotal weight of 15.0 liltres is yielded with the addition of water forinjectable preparations; 4) the resulting solution is then filteredthrough sterilizing filters and introduced in suitable sterile rooms(class 100) for its automatic ripartition in glass ampoules underatmosphere of nitrogen, using the techniques already well known in theart for the industrial manufacturing of injectable preparations.

[0147] After the chemical and biological (sterility and pyrogens)controls, the obtained ampoules are submitted to the usual automatictests in order to evidence foreign particles and the glass integritytests for ampoules, and then suitably packed individually in cartoonboxes for the distribution and its subsequent use in human medicine.1,421 ampoules of 10 ml (concentration of 50 mg/ml of MLc hydrochloride)are obtained with an effective yield of 94.73% (theoretical yield1,500).

[0148] Although the foregoing invention has been described in somedetail by way of illustration and examples, for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practised within the scope of the appended claims.

1) a compound of general formula:[CH₃O—(CH₂—CH₂—O)_(n)—CH₂—CH₂]_(x)—(NH)_(y)Lysozyme c. (Z)_(m) herein: yis the number of the free amino groups of lysine molecules present inthe molecule of lysozyme c; x is comprised between 1 and y; n iscomprised between 5 and 1000; Z is a pharmaceutically acceptable acid; mis comprised between 1 and y 2) A compound of general formula:[CH₃O—(CH₂—CH₂—O)_(n)—CH₂—CH₂]_(x)—(NH)_(y)Lysozyme c. (Z)_(m) wherein:y is the number of the free amino groups of lysine molecules present inthe molecule of lysozyme c; x is comprised between 1 and y; n iscomprised between 5 and 1000; Z is a pharmaceutically acceptable acid; mis comprised between 0 and y; with the proviso that, when lysozyme c ischicken egg-white lysozyme c, x is not 5 and n is not
 155. 3) A compoundaccording to claims 1-2 wherein n is comprised between 120 and
 200. 4) Acompound according to claims 1-2 wherein y is comprised between 5 and30. 5) A compound according to claims 1-2 wherein lysozyme c is fromchicken egg-white, rat, cow, rabbit, horse or human being. 6) A compoundaccording to claims 1-2 wherein said pharmaceutically acceptable acid ishydrochloric acid. 7) Use of a compound of formula[CH₃O—(CH₂—CH₂—O)_(n)—CH₂—CH₂]_(x)—(NH)_(y)Lysozyme c. (Z)_(m) wherein:y is the number of the free amino groups of lysine molecules present inthe molecule of lysozyme c; x is comprised between 1 and y; n iscomprised between 5 and 1000; Z is a pharmaceutically acceptable acid; mis comprised between 0 and y for the preparation of a pharmaceuticalcomposition to be administered by oral route able to induce highplasmatic levels of free TNF-α in mammalians. 8) Use according to claim7 for the preparation of a pharmaceutical composition to be administeredby oral route able to inhibit the replication speed of the tumoralproliferative process in mammalians. 9) Use of a compound of formula[CH₃O—(CH₂—CH₂—O)_(n)—CH₂—CH₂]_(x)—(NH)_(y)Lysozyme c. (Z)_(m) wherein:y is the number of the free amino groups of lysine molecules present inthe molecule of lysozyme c; x is comprised between 1 and y; n iscomprised between 5 and 1000; Z is a pharmaceutically acceptable acid; mis comprised between 0 and y for the preparation of a pharmaceuticalcomposition to be administered by parenteral route able to inhibit theplasmatic levels of TNF-α in mammalians. 10) Use according to claim 9for the preparation of a pharmaceutical composition to be administeredby parenteral route for the treatment and the prevention of the morbileconditions in mammalians, caused by TNF-α plasmatic levels higher thanphysiological normal values. 11) Use according to claims 9 and 10 forthe preparation of a pharmaceutical composition for the treatment andthe prevention of AIDS and its complications administrable by parenteralroute, preferably by intravenous route. 12) Use of a compound of formula[CH₃O—(CH₂—CH₂—O)_(n)—CH₂—CH₂]_(x)—(NH)_(y)Lysozyme c. (Z)_(m) wherein:y is the number of the free amino groups of lysine molecules present inthe molecule of lysozyme c; x is comprised between 1 and y; n iscomprised between 5 and 1000; Z is a pharmaceutically acceptable acid; mis comprised between 0 and y for the preparation of a pharmaceuticalcomposition administrable by topical route and/or intra-articular routeable to reduce in mammalians the concentration of TNF-α in the sinovialliquid. 13) Use according to claim 12 for the preparation of thepharmaceutical composition for the treatment of reactive arthritis andmore particularly of reumathoid arthritis. 14) Use of a compound offormula [CH₃O—(CH₂—CH₂—O)_(n)—CH₂—CH₂]_(x)—(NH)_(y)Lysozyme c. (Z)_(m)wherein: y is the number of the free amino groups of lysine moleculespresent in the molecule of lysozyme c; x is comprised between 1 and y, nis comprised between 5 and 1000; Z is a pharmaceutically acceptableacid; m is comprised between 0 and y for the preparation of apharmaceutical composition to be administered by topical route able toreduce in mammalians the local concentration of TNF-α in tissues. 15)Use according to claim 14, for the preparation of a pharmaceuticalcomposition with antihistaminic activity. 16) Use of a compound offormula [CH₃O—(CH₂—CH₂—O)_(n)—CH₂—CH₂]_(x)—(NH)_(y)Lysozyme c. (Z)_(m)wherein: y is the number of the free amino groups of lysine moleculespresent in the molecule of lysozyme c; x is comprised between 1 and y; nis comprised between 5 and 1000; Z is a pharmaceutically acceptableacid; m is comprised between 0 and y for the preparation of apharmaceutical compound for the treatment of psoriasis, of septic shockand of reumathoid arthritis in mammalians, to be administered byparenteral route, preferably by intravenous route. 17) Use according toclaims 7-16 wherein n is comprised between 120 and
 200. 18) Useaccording to claims 7-16 wherein y is comprised between 5 and
 30. 19)Use according to claims 7-16 wherein lysozyme c is from chickenegg-white, rat, cow, rabbit, horse or human being. 20) Use according toclaims 7-16 wherein said pharmaceutically acceptable acid ishydrochloric acid. 21) Pharmaceutical composition containing as activeingredient, a compound according to claims from 1 to 6, possibly incombination which one ore more different active ingredients, formulatedwith pharmaceutically acceptable ingredients and/or adjuvants. 22)Pharmaceutical composition according to claim 21, to be administered byoral route, containing from 5% to 80% by weight, preferably from 40% to60%, of the compound according to claims from 1 to
 5. 23) Pharmaceuticalcomposition according to claim 21, to be administered by parenteralroute, containing from 0.5% to 10% by weight, preferably from 2% to 4%,of the compound according to claims from 1 to
 5. 24) Pharmaceuticalcomposition according to claim 21, to be administered by topical route,containing from 0.5% to 10% by weight, preferably from 1% to 5%, of thecompound according to claims from 1 to
 5. 25) Manufacturing process forthe preparation of a compound according to claims from 1 to 6,comprising the following steps:
 1. monomethoxypolyethylene glycol isreacted with tresylated chloride.
 2. the resulting compound is reactedwith lysozyme c in a phosphate buffer saline or in a borate buffer in aslightly basic medium. 26) Manufacturing process according to claim 25,wherein the production of the addition salt is obtained via a dialysiswith an aqueous solution of acid Z, preferably for 6-8 hours at roomtemperature.